A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia

Shipra Bunjun, Constantinos Stathopoulos, David Graham, Bokkee Min, Makoto Kitabatake, Alice L. Wang, Ching C. Wang, Christian P. Vivarès, Louis M. Weiss, Dieter Söll

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coil tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(L-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Original languageEnglish (US)
Pages (from-to)12997-13002
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number24
DOIs
StatePublished - Nov 21 2000

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cysteinyl-tRNA synthetase
Amino Acyl-tRNA Synthetases
Giardia lamblia
Transfer RNA
Eukaryota
Giardia
Methanocaldococcus
Archaea
Enzymes
Adenosine Monophosphate
Escherichia coli
Genes
Alanine-tRNA Ligase
RNA, Transfer, Pro
Methanobacterium
prolyl T RNA synthetase
Proline
Adenosine
Saccharomyces cerevisiae
Organism Cloning

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia. / Bunjun, Shipra; Stathopoulos, Constantinos; Graham, David; Min, Bokkee; Kitabatake, Makoto; Wang, Alice L.; Wang, Ching C.; Vivarès, Christian P.; Weiss, Louis M.; Söll, Dieter.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 24, 21.11.2000, p. 12997-13002.

Research output: Contribution to journalArticle

Bunjun, S, Stathopoulos, C, Graham, D, Min, B, Kitabatake, M, Wang, AL, Wang, CC, Vivarès, CP, Weiss, LM & Söll, D 2000, 'A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia', Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 24, pp. 12997-13002. https://doi.org/10.1073/pnas.230444397
Bunjun, Shipra ; Stathopoulos, Constantinos ; Graham, David ; Min, Bokkee ; Kitabatake, Makoto ; Wang, Alice L. ; Wang, Ching C. ; Vivarès, Christian P. ; Weiss, Louis M. ; Söll, Dieter. / A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia. In: Proceedings of the National Academy of Sciences of the United States of America. 2000 ; Vol. 97, No. 24. pp. 12997-13002.
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abstract = "Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coil tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(L-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.",
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